Extended longevity is often correlated with increased resistance against the deleterious effects of environmental and physiological stresses, including heat and oxidative stresses. Our previous results suggested that the C. elegans heat-shock transcription factor (HSF-1), a master regulator of the cellular response to heat stress, is under direct regulation of the insulin/IGF-1-like signaling (IIS) networks to modulate the rate of aging and the onset of age-related diseases associated with proteotoxicity. The ultimate goal of our research is to develop new therapeutic strategies for combating age-related disease by understanding how HSF-1 influences the aging process. We have previously demonstrated that the rate of aging can be influenced by the level of HSF-1 activity in C. elegans, and that HSF-1 activity is required for daf-2 mutations to extend lifespan. Our recent findings suggest that IIS may control HSF-1 activity by regulating the formation of an inhibitory protein heterocomplex (DHIC) containing HSF-1, HSB-1, and two novel HSF-1 regulators, DDL-1 and -2. We found that the formation of DHIC is largely inhibited when IIS is reduced or when DDL-1 is phosphorylated at the T182 residue. We also found that the phosphorylation status of DDL-1 is regulated by IIS. Based on these observations, this proposal will focus on: 1) Further defining the role of DHIC complex in HSF-1 regulation. In this aim, we will develop a BiFC reporting system to verify the formation of DHIC in vivo and use it as a read-out to identify additional regulators of DHIC formation. We will also determine the structural requirements for the DHIC complex formation. 2) Determining the mechanism by which IIS regulates DDL-1 phosphorylation and DHIC formation. In the proposed studies, we will determine how the IIS pathway regulates DDL-1 phosphorylation and the formation of DHIC. 3) Identifying and characterizing additional regulators and effectors of HSF-1. Both heat-shock and reduction of IIS promotes an increase in the post- translational modifications (PTM) of HSF-1. In this aim, we will investigate the impacts of these PTMs on HSF- 1 activity. In addition, we will further characterize a number of novel HSF-1 regulators previously identified from a genetic screen.